Monoclonal antibody 2G12, isolated from HIV+ patient serum in 1996, neutralizes a broad range of HIV isolates and has been shown to be protective in animal models of HIV infection. 2G12 binds to a cluster of high-mannose (Man5-9GlcNAc2) glycans on HIV envelope protein gp120, and synthetic glycoclusters which closely mimic this epitope are of interest as immunogens which may be able to elicit a 2G12-like antibody response through vaccination.
There have been many attempts to design clusters of oligomannose glycans that mimic the 2G12 epitope. Chemical synthesis has enabled construction of well-defined structures in which glycans are mounted on numerous backbones, including cyclic peptides, PNA, dendrimers, and Qβ phage particles. Additionally, yeast strains have been engineered to express primarily high mannose carbohydrates on their surface. Unfortunately, none of these immunogens has been used successfully to raise a 2G12-like antibody response in vivo. In the best cases, when mannose-binding antibodies have been generated, their binding to gp120 or neutralization of HIV in vitro has still been weak or undetectable. Among several reasons for these failures is the likelihood that the clustering of oligomannose carbohydrates present in these immunogens did not sufficiently resemble the 2G12 epitope.
Optimized clustering of carbohydrates for more faithful mimicry of the 2G12 epitope was explored by using the antibody to recognize and select the best gp120 mimics from among a very diverse library. A new selection method, termed SELMA (SELection with Modified Aptamers), uses diverse DNA backbones to cluster the glycans in various ways (U.S. Patent Application Publ. No. 20130116417; MacPherson et al., Angew. Chem. Int. Ed. 50:11238-11242 (2011)). Libraries were constructed using copper assisted alkyne/azide cycloaddition (CuAAAC) chemistry to attach glycans to a library of random DNA sequences containing alkynyl bases. In single-stranded form, each DNA sequence clusters the glycans in a unique geometry, and the clusters that were selected from the library by binding to the target lectin (2G12 in this case) were amplified by PCR to generate a new library for further selection. The process was then repeated for several cycles with increasingly stringent selection conditions. By this method, clusters of 5-10 oligomannose glycans that were moderately good mimics of the 2G12 epitope were obtained; these constructs were recognized by 2G12 with 150-500 nM Kd's. However, the HIV envelope protein, gp120, is recognized much more tightly, with a Kd of ˜6-9 nM. To generate gp120 mimics that more faithfully replicate the glycan epitope, it will be necessary to generate glycan-oligonucleotides that are capable of binding to neutralizing monoclonal antibodies, like 2G12, with an affinity that is substantially the same as or less than that of the 120-2G12 interaction. Therefore, methods of developing better epitope mimics are needed.
The present invention is directed to overcoming these and other deficiencies in the art.